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University of Massachusetts Medical School University of Massachusetts Medical School

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GSBS Dissertations and Theses Graduate School of Biomedical Sciences

2012-08-06

Maintaining the Balance: Coordinating Excitation and Inhibition in

Maintaining the Balance: Coordinating Excitation and Inhibition in

a Simple Motor Circuit: A Dissertation

a Simple Motor Circuit: A Dissertation

Hilary A. Petrash

University of Massachusetts Medical School

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Petrash HA. (2012). Maintaining the Balance: Coordinating Excitation and Inhibition in a Simple Motor Circuit: A Dissertation. GSBS Dissertations and Theses. https://doi.org/10.13028/ptyx-an26. Retrieved from https://escholarship.umassmed.edu/gsbs_diss/633

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i

Maintaining the balance: coordinating excitation and inhibition in a

simple motor circuit

A Dissertation Presented By

Hilary A. Petrash

Submitted to the faculty of the

University of Massachusetts Graduate School of Biomedical Sciences, Worcester in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

August 6th, 2012

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ii

Maintaining the balance: coordinating excitation and inhibition in a simple motor circuit

A Dissertation Presented By

Hilary A. Petrash

The signatures of the Dissertation Defense Committee signifies completion and approval as to style and content of the Dissertation

Michael M. Francis, Thesis Advisor

___________________________________________________________________ Victor Ambros, Member of Committee

___________________________________________________________________ Claire Bénard, Member of Committee

___________________________________________________________________ Daniel Chase, Member of Committee

___________________________________________________________________ David Weaver, Member of Committee

The signature of the Chair of the Committee signifies that the written dissertation meets the requirements of the Dissertation Committee.

___________________________________________________________________ Mark Alkema, Chair of Committee

The signature of the Dean of the Graduate School of Biomedical Sciences signifies that the student has met all graduation requirements of the school.

___________________________________________________________________ Anthony Carruthers, Ph.D.,

Dean of the Graduate School of Biomedical Sciences Program in Neuroscience

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iii

Dedication

I would like to thank my parents, Howard and Doreen, for their support in my graduate school endeavor. Your support in my education and goals has been the foundation of my life’s success. I love you both. I would like to thank my sister, Merideth, who has provided comic relief during the long days of science. To the rest of my extended family, thank you for your everlasting encouragement.

To my husband, Don Petrash, you are a great joy in my life. My successes are sweeter when shared with you. You have given me so much patience and love during this journey and you never once doubted I would succeed. I love you and cannot thank you enough for believing in me. I look forward to the following chapters in our life together.

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iv

Acknowledgments

First and foremost, I would like to convey my deepest appreciation for my thesis advisor, Dr. Michael Francis, without whom I may never have learned to identify “the biological question”. You allowed me to work on a fascinating project that I have loved being involved in. Your support and encouragement have been critical to my success as a scientist. There are no words that I can think of to adequately thank you for the experience you have given me. I am truly grateful for the opportunity to have worked with you. Thank you.

I would like to thank the members of my Dissertation Defense Committee: Dr. Mark Alkema, Dr. Claire Bénard, Dr. David Weaver, Dr. Victor Ambros, and Dr. Daniel Chase for seeing me through the science and encouraging me to push ahead. Additionally, I would like to thank Dr. Scott Waddell and Dr. Heidi

Tissenbaum who provided me with encouragement and support in reaching my scientific goals.

To the faculty of the Neurobiology Department at UMass Medical School I would like to thank you for your feedback and guidance in my scientific

development. Lastly, I would like to thank all the members, past and present, of the Francis, Alkema, and Bénard laboratories for creating an environment that encourages discussions and development as a scientist. I have made lasting connections with many of these people, creating a second family that I hope to carry with me into the future. Thank you all.

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Abstract

The generation of complex behaviors often requires the coordinated activity of diverse sets of neural circuits in the brain. Activation of neuronal circuits drives behavior. Inappropriate signaling can contribute to cognitive disorders such as epilepsy, Parkinson’s, and addiction (Nordberg et al., 1992; Quik and McIntosh, 2006; Steinlein et al., 2012). The molecular mechanisms by which the activity of neural circuits is coordinated remain unclear. What are the molecules that regulate the timing of neural circuit activation and how is signaling between various neural circuits achieved? While much work has attempted to address these points, answers to these questions have been difficult to ascertain, in part owing to the diversity of molecules involved and the complex connectivity patterns of neural circuits in the mammalian brain.

My thesis work addresses these questions in the context of the nervous system of an invertebrate model organism, the nematode Caenorhabditis elegans. The locomotory circuit contains two subsets of motor neurons, excitatory and inhibitory, and the body wall muscle. Dyadic synapses from excitatory neurons coordinate the simultaneous activation of inhibitory neurons and body wall muscle. Here I identify a distinct class of ionotropic acetylcholine receptors (ACR-12R) that are expressed in GABA neurons and contain the subunit ACR-12. ACR-12R localize to synapses of GABA neurons and facilitate consistent body bend amplitude across consecutive body bends. ACR-12Rs regulate GABA neuron activity under conditions of elevated ACh release. This is

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vi in contrast to the diffuse and modulatory role of ACR-12 containing receptors expressed in cholinergic motor neurons (ACR-2R) (Barbagallo et al., 2010; Jospin et al., 2009). Additionally, I show transgenic animals expressing ACR-12 with a mutation in the second transmembrane domain [ACR-12(V/S)] results in spontaneous contractions. Unexpectedly, I found expression of ACR-12(V/S) results in the preferential toxicity of GABA neurons. Interestingly loss of

presynaptic GABA neurons did not have any obvious effects on inhibitory NMJ receptor localization. Together, my thesis work demonstrates the diverse roles of nicotinic acetylcholine receptors (nAChRs) in the regulation of neuronal activity that underlies nematode movement. The findings presented here are broadly applicable to the mechanisms of cholinergic signaling in vertebrate models.

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Table of Contents

Cover Page i Signature Page ii Dedication iii Acknowledgments iv Abstract v

Table of Contents vii

List of Tables ix

List of Figures x

List of Third Party Copyright xii

List of Abbreviations of Nomenclature xiii

Preface xv

Chapter I: Introduction 1

Chapter II: Excitation and inhibition are coordinated through multiple cholinergic (nicotinic) signaling pathways in C. elegans motor control 28

Chapter III: A sensitized nAChR leads to spontaneous contractions and GABA neurons toxicity in the C. elegans locomotory circuit 66

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viii

Table of Contents

Appendix I: A Dominant Mutation in a Neuronal Acetylcholine Receptor

Subunit Leads to Motor Neuron Degeneration in Caenorhabditis elegans 112

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ix

List of Tables

Table A1-1. Several nAChR subunits are required for ACR-2(L/S)-induced

paralysis

Table A1-S1. Suppressors of ACR-2(L/S) induced paralysis isolated from a

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x

List of Figures

Figure 1-1 (Doyle, 2004). Crystal structure of nicotinic acetylcholine receptor

side and top view

Figure 1-2 (Revah et al., 1991). Diagram of TM2 of nAChRs with important

residues indicated along with functional kinetics data

Figure 1-3 (Jones and Sattelle, 2004). Phylogeny of C. elegans nAChR

subunits aligned with vertebrate nAChRs, and other members of LGICs

Figure 1-4 (Altun, 2011). Diagram of locomotory connectivity in C. elegans.

Figure 2-1 ACR-12 gene sequence.

Figure 2-2 ACR-12 iAChRs regulate GABA motor neuron activity

Figure 2-3 ACR-12 is differentially localized across motor neuron populations

Figure 2-4 ACR-12 is localized at synapses on GABA motor neurons

Figure 2-5 Loss of ACR-12 receptors reduces inhibitory signaling

Figure 2-6 ACR-12 receptors are required for consistent motor patterning

Figure 2-7 GABA-specific ACR-12 receptors mediate elevated levels ACh

signaling.

Figure 2-8 GABA-specific ACR-12 receptors are important under conditions of

elevated ACh release

Figure 3-1 Mutation in a conserved residue of ACR-12

Figure 3-2 ACR-12(V/S) animals exhibit dramatic behavioral deficits

Figure 3-3 Expression of ACR-12(V/S) is toxic in GABA motor neurons

Figure 3-3 ACR-12(V/S) toxicity is observable in L1 larva

Figure 3-5 GABA-specific expression of ACR-12(V/S)

Figure A1-1. acr-2 is expressed in cholinergic motor neurons and modulates

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xi

Figure A1-2. Transgenic animals expressing the dominant ACR-2(L/S)

transgene are severely uncoordinated.

Figure A1-3. Transgenic expression of ACR-2(L/S) leads to a loss of cholinergic

motor neurons.

Figure A1-4. ACR-2(L/S)-induced motor neuron cell death is initiated before

hatch.

Figure A1-5. Mutations in nicotinic acetylcholine receptor subunits suppress

ACR-2(L/S)-induced paralysis.

Figure A1-6. ACR-2(L/S)-mediated motor neuron loss is completely prevented in

animals doubly mutant for calnexin and calreticulin.

Figure A1-7. Progressive destabilization of motor neuron processes in

cnx-1;crt-1;ACR-2(L/S) animals.

Figure A1-8. Perturbation of intracellular calcium and reduced ACR-2::GFP

levels in cnx-1;crt-1 double mutants suggest dual mechanisms for cell death suppression.

Figure A1-S1. Expression of the non-alpha nAChR subunit ACR-2.

Figure A1-S2. C. elegans command interneurons do not express Punc-17::GFP.

Figure A1-S3. Sequence features of ACR-2.

Figure A1-S4. Wild type and ACR-2(L/S) animals have the same number of

acr-2 expressing cells at the 3-fold embryo stage.

Figure A1-S5. The VC neurons remain present in transgenic ACR-2(L/S)

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xii

List of Third Party Copyrighted Material

Figure Number Figure 1-2 Figure 1-3 Figure 1-4 Publisher License Elsevier Elsevier

John Wiley and Sons

Number

2911040177191 2913730090430 2913830200516

The following figures were reproduced from journals or books: no permission required

Figure Number

Figure 1-5

Publisher

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xiii

List of Abbreviations or Nomenclature

ACh- acetylcholine

AChR- acetylcholine receptor

ACR- acetylcholine receptor (C. elegans specific)

ADNFLE- autosomal dominant nocturnal frontal lobe epilepsy ALS- Amyotrophic lateral sclerosis

CNS- central nervous system ER- endoplasmic reticulum GABA- γ-amino butyric acid

iAChRs- ionotrophic acetylcholine receptors ICL- intracellular loop

iGluRs- ionotrophic glutamate receptors MNs- motor neurons

NAc- nucleus accumbens

nAChR- nicotinic acetylcholine receptor NMJ- neuromuscular junction

L-AChRs- levamisole sensitive AChR LDT- laterodorsal tegmentum

LGIC- ligand gated ion channel PSC- postsynaptic current TM- transmembrane

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xiv *- containing (ex. α7* means α7 containing)

A motor neurons- backward movement B motor neurons- forward movement

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xv

Preface

All work described in this thesis was performed at University of Massachusetts Medical School in the lab of Michael M. Francis.

In chapter II, Alison Philbrook built the cholinergic motor neuron specific channelrhodopsin in the acr-2 mutant and cholinergic channelrhodopsin in the acr-12 mutant and helped perform the channelrhodopsin experiments. Marian Habrucak contributed to the electrophysiology. Michael M. Francis supported this work in several ways including discussions, designing the experiments, writing the paper, and contributing the electrophysiological experiments. I designed and performed the remaining experiments, analyzed the data, and co-wrote the paper.

In chapter III, Michael M. Francis supported this work with discussions and designing the experiments. I designed and performed the experiments and

analyzed the data.

In appendix I, I contributed cell specific rescue strains of acr-12 for identifying neurons sufficient for ufIs25 paralysis, strain building, and

coexpression of cholinergic and GABAergic neurons. The remaining authors were responsible for the rest of the work.

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1

Chapter 1:

Introduction

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2 The nervous system is a complex and highly interconnected

network of specialized cells called neurons. Groups of connecting neurons form circuits that modify behavior based on activity. These neurons are interconnected by two mechanisms: the electrical synapse, or gap junction, and a unique

chemical junction called a synapse. Electrical coupling or gap junctions are a direct low resistance mechanism for transmitting electrical activity from one neuron to a neighboring neuron or other cell type. The gap junction is comprised of a hydrophilic intercellular channel, with one subunit of the channel contributed from each of the connecting cells (Flores et al., 2012; Pereda et al., 2012). In contrast, synapses utilize chemical transmission. An electrical signal from the presynapse triggers the release of a neurotransmitter into the synaptic cleft. After diffusing across the cleft, the neurotransmitter binds to receptors located on the postsynaptic neuron. Fast synaptic transmission is mediated by ionotropic receptors, also known as ligand-gated ion channels (LGICs). Binding of the chemical neurotransmitter triggers a conformational change in the receptor, opening a pore through the membrane that allows the flow of ions into or out of the cell, and converting the chemical signal into an electrical response (Holtmaat and Svoboda, 2009). Chemical signaling through a synapse translates into one of two simple outcomes: (1) the activation of the postsynaptic neuron and continued neuronal transmission or (2) the inhibition of the postsynaptic neuron that reduces downstream signaling. Signaling at synapses occurs on a time scale of milliseconds, allowing for rapid activation or inhibition of sequential neurons

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3 within a circuit to appropriately drive circuit activity and ultimately determine behavior.

The generation of complex behaviors often requires the coordinated activity of diverse sets of neural circuits in the brain. The molecular mechanisms by which the activity of neural circuits is coordinated remain unclear. What are the molecules that regulate the timing of neural circuit activation and how is signaling between various neural circuits achieved? These are important questions, as disruption of coordinated circuit activity can have severe consequences (e.g., epilepsy). While scientific research has attempted to

address these points, answers to these questions have been difficult to ascertain, in part owing to the diversity of molecules involved and the complex connectivity patterns of neural circuits in the mammalian brain. My thesis work addresses these questions in the context of the nervous system of an invertebrate model organism, the nematode Caenorhabditis elegans. The genetic tools available and well-established connectivity of this organism make it ideal for this type of study. As the major neurotransmitter in C. elegans is acetylcholine, a detailed

description of cholinergic signaling and the molecules involved follows. Ligand-gated ion channels and synaptic transmission

The ligand-gated gated ion channel (LGIC) super family includes receptors for the neurotransmitters glutamate, acetylcholine (ACh), γ amino butyric acid (GABA), glycine, and a single serotonin receptor (Corringer et al.,

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4 2012; Millar and Harkness, 2008; Rosenberg et al., 2002). In mammals, LGIC family members can be broadly classified as either excitatory or inhibitory based on their permeability to either anions or cations. Ionotropic glutamate receptors (iGluR) and nicotinic acetylcholine receptors (nAChR) act as cation-selective channels and are excitatory, while GABA and glycine receptors act as anion-selective channels and are generally inhibitory in the adult nervous system. Over 40 distinct genes that encode subunits of LGICs have been identified in

mammals (Baenziger and Corringer, 2011). These receptors are pentameric, with the exception of glutamate receptors which form tetramers, and are

expressed in varying subunit combinations throughout the nervous system (Millar and Gotti, 2009). Pharmaceutical therapies designed to activate (agonists) or inhibit (antagonists) specific subunit combinations have been developed as potential treatments for diseases such as epilepsy and Parkinson’s (Brooks-Kayal et al., 1998; Davies, 1995; Mulley et al., 2005; Quik and McIntosh, 2006). In Parkinson’s disease stimulation of presynaptic nAChRs can increase

dopamine release in the substania nigra (Quik and McIntosh, 2006). Current treatments for Parkinson’s disease synthetically compensate for a loss of dopamine using L-DOPA, a precursor of dopamine. However, as previously mentioned in Parkinson’s models it is possible to achieve similar increases in dopamine levels by activating presynaptic nAChRs (Quik and McIntosh, 2006). nAChRs were the first members of the LGIC family to be isolated and studied in

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5 vivo and much of our understanding of how LGICs function has come from

studies of nAChRs (Unwin, 2005).

Structure, function and localization of nicotinic acetylcholine receptors

nAChRs were first isolated from the electric organ of the marine Torpedo ray and the Electrophorus eel (Changeux et al., 1970; Popot and Changeux, 1984). In vertebrates, there are 17 nAChR subunits: α1-10, β1-4, δ, γ, and ε (Lindstrom, 2003; Millar and Harkness, 2008). In general, nAChRs fall into two classes, receptors comprised of identical subunits (homomeric) or different subunits types (heteromeric) (Unwin, 2005).The heteromeric nAChR of the NMJ contains the subunits α12β1δε; although, during development the subunit

composition is slightly different (a γ subunit is substituted for an ε subunit) (Millar and Harkness, 2008). In the nervous system the subunit composition of nAChRs has more potential for variability, enabling functional diversity across specific subunit combinations. Receptors formed from the alpha7 subunit (homologous to the ACR-16 subunit in C. elegans) are generally homomeric while the other subunits typically contribute to heteromeric receptors, the precise subunit combinations of which vary across neuronal classes.

Each subunit of the pentameric nAChR contains four membrane spanning domains (Lindstrom, 2003). Activation of the channel occurs when ACh attaches to a binding site on the external face of the receptor. Initially, the extracellular N-terminal region of α subunits was thought to be solely responsible for ligand

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6 binding. However, more recent studies have shown that the ACh binding sites exist at the interface of adjacent subunits (Corringer et al., 2000; Luetje and Patrick, 1991). Proper formation of the ACh binding site is dependent on the presence of two adjacent cysteines located in the N-terminal region of α subunits (Figure 1-1) (Doyle, 2004; Lindstrom, 2003). Subunits without these adjacent cysteines are classified as non-alpha: β, δ, γ, and ε subunits. Each subunit has a large intracellular loop between the third and fourth transmembrane domains. The sequence of this intracellular loop does not appear to be highly conserved across other nAChR subunits of the same or different species (Doyle, 2004; Lindstrom, 2003). It is likely that sequence elements within the large intracellular loop are important for localization of the receptor.

The second transmembrane domain of each subunit contributes to the ion channel pore of the receptor (Imoto et al., 1988; Revah et al., 1991). Mutations in specific residues of the pore region and the area directly surrounding the pore can dramatically alter kinetics, prolonging channel open time (Figure 1-2). For example, mutations at amino acids 234, 237 and 258 convert a channel from cationic to anionic. Residues 234 and 237 are located just outside the second transmembrane domain in the extracellular space while residue 258 is located at the intracellular end of the second transmembrane domain (Revah et al., 1991). Others have identified residues 236 and 251 as contributing to ion selectivity (Corringer et al., 1999; Galzi et al., 1992; Imoto et al., 1988). Reconstitution studies have shown the kinetics of α7 homomers could be dramatically altered by

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7 mutating a residue in the second transmembrane domain. Mutating residue 247 from a non-polar leucine to a polar serine has the most pronounced effect on mean channel open time (Revah et al., 1991). Hypersensitive nAChRs, or gain of function receptors, have since been used to examine the role of cholinergic signaling in the CNS. Using an α4 gain of function knock in mouse model, researchers were able to identify interactions of nAChRs and dopamine D2

receptors in regulating cholinergic interneuron activity (Zhao-Shea et al., 2010). Mutations in the pore lining domain of α6 (α6L9’S

) nAChR subunit identified α6α4β2* containing receptors as a key target for disorders associated with reduced dopamine release. The researchers were able to specifically link the behavioral deficits of α6L9’S animals to α6α4β2* containing receptors in dopamine

releasing neurons in vivo (Drenan et al., 2010). Additional work with gain of function α6 subunits has demonstrated a role for β3 subunits in promoting the function of α6 containing receptors (Dash and Lukas, 2012).

Role of cholinergic signaling in the nervous system

In the CNS nAChRs are predominantly localized at presynaptic terminals, while postsynaptic nAChRs act in the peripheral nervous system to mediate transmission in the sympathetic ganglia (Rassadi et al., 2005). Cholinergic

signaling coordinates neuronal activity in several manners. First, nAChRs located at presynaptic sites are important in modulating the release of neurotransmitter. For example, activation of presynaptic nAChRs on dopamine neurons increases

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8 neurotransmitter release (Salminen et al., 2004). Second, activation of

pre-synaptic nAChRs can trigger neurotransmitter release in the absence of a

propagated signal; bypassing upstream signaling (Vizi and Lendvai, 1999). Third, cholinergic signaling is linked to changes in gene expression (Chang and Berg, 2001; Hu et al., 2002). Researchers have demonstrated that selective activation of nAChRs leads to the phosphroylation of CREB, a transcriptional coactivator, and changes in expression of the immediate early gene c-Fos. Fourth, nAChRs can directly activate neurons by localizing to postsynaptic sites. It is possible subunit diversity in nAChRs provides an opportunity for diverse roles for cholinergic signaling.

Altered cholinergic signaling is implicated in the pathophysiology of epilepsy, Parkinson’s disease, Alzheimer’s disease, and in addiction to nicotine (Dineley, 2007; Gotti and Clementi, 2004; Quik and McIntosh, 2006; Steinlein et al., 2012). Activation of nAChRs in the reward pathways of the brain facilitates nicotine addiction. There are at least three distinct classes of nAChRs involved in nicotine addiction α4*, α6*, and α7. Specifically, nicotine binds to presynaptic α7 receptors on glutamatergic neurons in the laterodorsal tegmentum (LTD)

influencing neurotransmitter release onto dopaminergic neurons in the ventral tegmentum area (VTA). The dopaminergic neurons express two classes of nAChRs, α4β2*- and β2*- containing receptors, which modulate neurotransmitter release and increase in burst firing (Dani et al., 2001; Mansvelder and McGehee, 2002). Inhibitory GABA neurons also make synaptic contacts with dopaminergic

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9 neurons of the VTA and express α4β2 containing receptors (Dani and Harris, 2005). However, distinct nAChRs extend beyond the reward pathways of the brain. α3β4 receptors were recently identified as directly mediating excitation of mitral cells from olfactory inputs. Activation of mitral cells filters the inputs from olfactory neurons (D'Souza and Vijayaraghavan, 2012). Additionally, impaired cholinergic signaling in Alzheimer’s disease results in decreases in cognition (McGehee et al., 1995). Mutations in α4 and β2 subunits have been linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) (Raggenbass and Bertrand, 2002). There are age and dementia associated changes in high and low affinity nAChRs in various regions of the brain (Nordberg et al., 1992). During development, loss of α7 containing receptors dramatically reduces the number of glutamatergic synapses in the adult brain. The reduction in glutamatergic

synapses is associated with the behavioral changes in α7 knockout mice including attention deficits and impaired spatial discrimination (Lozada et al., 2012).

C. elegans as a model for studying nervous system function

C. elegans is an ideal invertebrate model for addressing fundamental principles of nervous system function. There are a host of cell specific drivers and the cell fate of all embryonic cells is known (Hobert, 2005). The genome of the organism has been fully sequenced and there is a myriad of genetic tools available, as well as readily available deletion mutants for many genes of

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10 interest. Under laboratory conditions, C. elegans strains are maintained with a readily available bacterial food source so the animals do not have to move in order to feed. In addition, C. elegans are hermaphroditic so coordinated motor behaviors are not required for reproduction. These features allow one to propagate strains carrying severe mutations in genes required for nervous system function that would be lethal in most systems, and study the functional roles of these genes. In addition, the transparent body enables in vivo

visualization of fluorescent markers in the intact animal. More recently developed techniques have made C. elegans amenable to electrophysiological experiments, which can be performed both in vivo and in vitro (Christensen et al., 2002;

Francis et al., 2003; Richmond and Jorgensen, 1999). In addition, tools such as cell specific expression of light-activated ion channels, Channelrhodopsin-2 or Halorhodopsin, allow for the targeted activation of specific neuronal circuits in vivo and subsequent analysis using behavior and electrophysiology (Nagel et al., 2005; Zhang et al., 2007a). Qualities of the animal’s characteristic sinusoidal movement can be measured under a variety of conditions. These tools allow researchers to tease apart how a single neuronal circuit contributes to a specific behavior.

The C. elegans nervous system uses many of the same neurotransmitters as mammals including acetylcholine, GABA, and glutamate. The genome is about 97 megabases and contains over 19,000 genes (Consortium, 1998). This genome has a high density of receptors important for synaptic function. In

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11 addition, there are on the order of 90 neurotransmitter-gated ion channels of which there are more than 30 potential nAChR subunits (Bargmann, 1998; Jones et al., 2007; Jones and Sattelle, 2004). C. elegans possess the largest known family of AChRs (Mongan et al., 2002). These subunits fall into six different groups named for the first subunit identified in each group: unc-29, unc-38, acr-8, acr-16, deg-3, and orphan LGIC (Figure 1-3). Specifically, the acr-8 and deg-3 groups are unique to C. elegans (Jones and Sattelle, 2004). The acr-8 group has a unique feature compared to the other groups with a basic (histidine) residue in place of a highly conserve acidic (glutamate) residue in the second

transmembrane domain of the receptor. This alteration can alter the ion

selectivity from cation to anion (Corringer et al., 1999; Imoto et al., 1988). The C. elegans nAChR subunits are classified as either α (ligand binding) or non-α (non-ligand binding) by the absence or presence of adjacent cysteines. Similar to mammalian subunits, C. elegans nAChRs show differential expression patterns and are not limited to a specific neuron class (Barbagallo et al., 2010; Cinar et al., 2005; Drenan et al., 2008; Fox et al., 2005; Jospin et al., 2009; Nordberg et al., 1992; Salminen et al., 2004).

Genes involved in the trafficking of AChRs include Calnexin, BiP, unc-50, ric-3, and unc-74 (Boulin et al., 2008; Eimer et al., 2007; Forsayeth et al., 1992; Gelman et al., 1995). Both Calnexin and Bip are ER chaperones involved in the synthesis of nAChRs and are shed in the maturation process (Forsayeth et al., 1992; Gelman et al., 1995). Unfolded or misassembled subunits are retained in

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12 the ER and degraded by ER-associated degradation (Christianson and Green, 2004). UNC-50 is specifically required for the trafficking of levamisole sensitive AChRs (L-AChRs). These proteins are involved in subunit specific trafficking of nAChRs, while the function of unc-74 remains unknown (Eimer et al., 2007). RIC-3 is a protein important for the trafficking of nAChR, but not other LGIC, to the cell membrane and is localized to the endoplasmic reticulum (ER) (Halevi et al., 2002). This is similar to the human form of ric-3 (hric3); however, the human form has further evolved to inhibit certain subunit combinations of nAChR and extend its regulation to other LGIC (Halevi et al., 2003). Taken together this evidence suggests factors involved in trafficking and localization are key regulators in preventing aberrant receptors from reaching the cell membrane. In addition, there are functional properties associated with distinct subunit compositions such as agonist sensitivity and calcium permeability (Changeux et al., 1970; Deneris et al., 1991; Drisdel and Green, 2000; Lipovsek et al., 2012). The C. elegans

locomotory circuit is enriched for nAChRs making this circuit of alternating movement ideal for examining coordinated activity.

Generating alternating movements in C. elegans

Utilizing alternating movements as a model for identifying mechanisms that coordinate excitation and inhibition is common (Eklof-Ljunggren et al., 2012; Gabriel et al., 2011; Grillner and Jessell, 2009; Kupfermann and Weiss, 2001; Masino and Fetcho, 2005). One of the primary advantages to this model is a

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13 completely defined network of neurons including the anatomical connections of all chemical and electrical synapses (White et al., 1986). The locomotory circuit is comprised of command interneurons, excitatory, and inhibitory motor neurons. Sensory information is relayed through command interneurons that synapse onto excitatory motor neurons. There are 5 sets of command interneurons: AVA, AVB, AVE, AVD, and PVC (White et al., 1986). AVA, AVE, and AVD regulate

backwards movement through electrical and chemical synapses with the A motor neurons (backwards movement). AVB and PVC regulate forward movement via gap junctions and chemical synapses with B type motor neurons (forward movement) (Chalfie et al., 1985; Wicks et al., 1996; Zheng et al., 1999). Recent work has suggested gap junctions between AVA and A motor neurons function to bias the motor circuit towards forward versus backward movement. In addition electrical coupling may be modulatory in forward movement or there are

alternative signaling mechanisms maintaining the communication between AVB and B type motor neurons in forward movement (Kawano et al., 2011). In turn, the excitatory motor neurons (A and B), form dyadic synapses with body wall muscle (BWM) initiating contraction while simultaneously activating inhibitory motor neurons. Activation of the inhibitory motor neurons initiates relaxation of the contralateral BWM. The simultaneous signaling of excitation and inhibition on opposing muscle groups is thought to be important for generation of the animal’s characteristic sinusoidal wave form (Figure 1-4) (White et al., 1986).

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14 In vertebrates there are fundamental differences in NMJ and CNS

synapses. First NMJ synapses are far more stable than those of the CNS which have a higher degree of plasticity. During development, the half life of receptors at the NMJ are lengthened unlike the receptors of the CNS. Second, the

formation of CNS synapses occurs on the time scale of hours whereas the time scale at the NMJ is weeks (Holtmaat and Svoboda, 2009). Third, proteins

associated with NMJ formation (i.e. Agrin, MuSK, and LRP4) are not required for CNS synapse formation. Lastly, in stark contrast to the CNS synapses the NMJ is regulated primarily by one neurotransmitter and one class of nAChR

(Lindstrom, 2003). In the CNS neurons are polyinnervated, receiving both

inhibitory and excitatory neurotransmitters (Kim et al., 2008; Zhang et al., 2008). The C. elegans NMJ is similar in that it is also polyinnervated, expresses different classes of receptors, and forms through mechanisms independent of Agrin, MuSK, and LRP4 (Francis et al., 2005; Richmond and Jorgensen, 1999;

Touroutine et al., 2005) . Therefore, identifying molecular mechanisms involved in regulating the activity of the C. elegans locomotory circuit will be relevant for linking findings to mechanisms in the mammalian CNS.

Within the C. elegans locomotory circuit are previously characterized nAChRs. One receptor is a homomeric (contains one subunit type) receptor containing five Acetylcholine Receptor-16 (ACR-16) (Francis et al., 2005; Touroutine et al., 2005) subunits, and a second receptor is heteromeric

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15 al., 2004; Fleming et al., 1997; Unwin, 2005). The 29, 38, and UNC-63 subunits are all essential for the function of the UNC-29 receptor. LEV-1 and LEV-8 are considered non-essential subunits. Similar to mammalian CNS synapses the inhibitory NMJ is mediated by a GABA receptor (UNC-49). This is in contrast to the mammalian NMJ which is regulated solely by nAChR.

Formation of the C. elegans NMJ requires proteins such as CAM-1, a receptor tyrosine kinase, responsible for the proper trafficking of ACR-16 receptors (Fleming et al., 1997). CAM-1 is orthologus to the mammalian ROR1 and 2 (Francis et al., 2005; Jensen et al., 2012). Furthermore, LEV-10 is a single pass transmembrane protein with several CUB domains which has been implicated in receptor clustering at the C. elegans NMJ (Gally et al., 2004). The mechanisms introduced here generate the characteristic alternating movement of C. elegans. The locomotory circuit of C. elegans is one of many models of alternating

movement being utilized for understanding nervous system function.

Alternating movement models in characterizing mechanisms of neuronal activity

Walking vertebrates or swimming fish are classic demonstrations of the precision required to maintain the balance of excitatory and inhibitory signaling in movement circuits. Alternating movement can be seen in a host of organisms ranging from humans to C. elegans. Opposing muscle groups coordinate their activation and relaxation in order to properly time the shortening of one group of muscles while simultaneously lengthening the contralateral muscle group.

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16 Alternating movements are finely modulated to achieve a variety of speeds, direction, amplitude, and frequency. Similar to the neuronal circuits in the brain, alternating movements require molecular mechanisms to facilitate adaptation to stimuli with a high degree of precision while maintaining a coordinated balance of excitation and inhibition. The molecular mechanisms that regulate activity in alternating movements are easier to study than those of the mammalian central nervous system. This is due in part to the well defined circuitry providing a more controlled environment for identifying and characterizing mechanisms of neuronal activity. This model circuit is an ideal alternative to the complex neuronal circuitry of the mammalian brain.

Motor circuits have been characterized in several model systems including lamprey, zebrafish, mouse, and nematode (Goulding, 2009). The lamprey and zebrafish model systems have a unique feature of maintaining the complexity of highly connected interneuron populations while limiting the density of neurons (Eklof-Ljunggren et al., 2012; Masino and Fetcho, 2005). Comparing interneuron populations across species makes circuit analysis in vertebrate models more broadly relevant (Fetcho et al., 2008). Vertebrates allow us to address questions of how specific subsets of interneurons or motor neurons affect alternating movements and what kinds of molecular mechanisms are involved. However, invertebrate models present their own advantages for probing locomotory circuits. First, the circuitry is vastly simplified compared to vertebrate models (Altun, 2008; Jing and Weiss, 2002; Kupfermann and Weiss, 2001). Second, as

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17 previously mentioned the connectivity of all neurons and synapses are identified and well characterized (Serrano et al., 2007; White et al., 1986). Third, the generation time of some invertebrate model organisms profoundly shorter than the generation time of vertebrate models. For example, the generation time of C. elegans is only 4 days. This allows researchers to perform large forward genetic screens and isolate mutants much faster than that of alternative model systems. Ultimately, the C. elegans locomotory circuit is ideally suited for linking

mechanisms of neuronal activity to those of the mammalian CNS. Dissertation Overview

My thesis work will focus on the chemical synapse, specifically how receptors regulate neuronal activity and directly influence behavior. I use the locomotory circuit of C. elegans as a model for identifying mechanisms that coordinate neuronal activity. The molecular mechanisms are critical to the balance of excitation and inhibition. The elucidation of these mechanisms will provide insights into mammalian nervous system function. In order to understand and study nervous system function it is important to be familiar with the types of molecular mechanisms that affect neuronal activity. Here in the first chapter I have introduced the structure and function of the nervous system including both chemical synapses and electrical coupling. I have also provided background on mechanisms that regulate neuronal activity and specifically the role of cholinergic

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18 signaling in regulating neuronal activity. I have ended with establishing C.

elegans as an informative model for studying nervous system function. Chapter II identifies a mechanism important in directly coordinating excitatory and inhibitory signaling in the C. elegans locomotory circuit. Here, I provide evidence that a single nAChR subunit, ACR-12, forms two distinct classes of nAChRs. These distinct classes have differential localization, composition, and roles in regulating behavior. Our previous work with acr-2 identified a heteromeric nAChR expressed in cholinergic motor neurons (ACR-2R) (Barbagallo et al., 2010). The ACR-2R has diffuse localization in the dendrites and a modulatory role in regulating neuron activity (Barbagallo et al., 2010; Jospin et al., 2009). Loss of ACR-2Rs results in subtle changes in behavior (see Appendix). In contrast the recently identified GABA neuron-specific ACR-12 (ACR-12R) localizes to discrete sites within the dendrites. Colocalization of these discrete sites opposed to a presynaptic reporter suggests ACR-12R is synaptic. Since two of the subunits from the ACR-2R are not expressed in GABA neurons, this observation suggests that GABA neuron-specific ACR-12 containing

receptors are distinct from the heteromeric cholinergic receptor. In addition, the ACR-12R is implicated in regulating the consistency of the C. elegans waveform and mediating the activity of GABA neurons under conditions of elevated

acetylcholine (ACh) release. Taken together, these results suggest diverse roles for receptor subtypes are important for coordinating neuronal activity.

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19 Chapter III identifies the contributions of neuronal subsets to the

generation of movement. Data in this chapter demonstrates that expression of a gain of function ACR-12 [ACR-12(V/S)] receptor has dramatic effects on behavior including highly reduced movement and spontaneous convulsions. In addition, expression of this transgene leads to the degeneration of the GABAergic nervous system, but not the cholinergic nervous system. These degenerative effects can be observed at early life stages and preliminary evidence suggests this

degeneration is cell autonomous. Interestingly, the NMJ inhibitory receptor, UNC-49, remains unaffected by the degeneration of presynaptic GABA neurons. Our results indicate ACR-12(V/S) will be a useful tool in identifying how subsets of neurons contribute to the overall behavior of an animal. Lastly, expression of ACR-12(V/S) may prove a useful genetic tool for identifying proteins associated with receptor localization, subunit identification, and mechanisms of

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20

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21

Figure 1-1 reprint from (Doyle, 2004). The structure of a nAChR. (a) An extracellular perspective of the transmembrane helical topology in its

closed state. (b). A side view (γ subunit removed). For all subunits (2α, β, and δ), transmembrane (TM) TM1 (red), TM2 (magenta), TM3 (green) and TM4 (blue). Residues of the hydrophobic gate are modeled in yellow ball-and-stick. TM2 rotation direction is indicated by the arrows.

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22

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23

Figure 1-2 reprint from (Revah et al., 1991): “b, Functional properties of α7

receptor mutants. b, Normalized currents evoked by 100μM ACh in Xenopus oocytes expressing α7 wild type (WT) and α7 L247F, L247V, L247T, L247S, L246T, S248A receptors (where letters represent amino acids at positions given by the numbers) are superimposed. The normalized responses to ACh from WT, L246T and S248A were indistinguishable.”

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24

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25

Figure 1-3 (Jones and Sattelle, 2004): Phylogeny of C. elegans nAChR

subunits with vertebrate AChR subunits and other members of the LGIC superfamily. C. elegans α subunits (blue), non-α subunits (red), and GABA, glycine, glutamate, 5-HT3 and vertebrate nAChR subunits (black). Cosmid numbers are used for C. elegans sequences lacking gene names.

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26 Figure 1-4

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27

Figure 1-4 (Altun, 2011). Schematic of C. elegans locomotory circuit. Dark and

light green shading indicates body wall muscles. In blue are the ACh MNs (VA, VB, DA, DB, AS, VC) and orange indicates GABA MNs (VD and DD). Axons and dendrites of ventrally directed cholinergic motor neurons (ventral A and ventral B classes) extend through the ventral nerve cord. Dorsally directed cholinergic motor neuron cell bodies (dorsal A class and dorsal B classes) are located adjacent to the ventral nerve cord and extend axons into the dorsal nerve cord, where they make dyadic synaptic contacts with body wall musculature and dendrites of ventrally directed GABA motor neurons (ventral D class).

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28

Chapter II:

Excitation and inhibition are coordinated

through multiple cholinergic (nicotinic)

signaling pathways in C. elegans motor

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29

ABSTRACT

Heterogeneity in the composition of neurotransmitter receptors is thought to provide functional diversity that may be important in patterning neural activity and shaping behavior (Dani and Bertrand, 2007; Sassoe-Pognetto, 2011). However, this idea has remained difficult to evaluate directly due to the complexity of neuronal connectivity patterns and uncertainty about the molecular composition of specific receptor types in vivo. Here we dissect how molecular diversity across receptor types contributes to the coordinated activity of excitatory and inhibitory motor neurons in the nematode Caenorhabditis elegans. We show that excitatory and inhibitory motor neurons express distinct populations of ionotropic

acetylcholine receptors (iAChR) requiring the ACR-12 subunit. The activity level of excitatory motor neurons is influenced through activation of nonsynaptic iAChRs (Barbagallo et al., 2010; Jospin et al., 2009). In contrast, synaptic

coupling of excitatory and inhibitory motor neurons is achieved through a second population of iAChRs specifically localized at postsynaptic sites on inhibitory motor neurons. Loss of ACR-12 iAChRs from inhibitory motor neurons leads to reduced synaptic drive, decreased inhibitory neuromuscular signaling and variability in the sinusoidal motor pattern. Our results provide new insights into mechanisms that establish appropriately balanced excitation and inhibition in the generation of a rhythmic motor behavior, and reveal functionally diverse roles for iAChR mediated signaling in this process.

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30

INTRODUCTION

The pattern of activity in neuronal circuits is the basis for behavior, addition, learning and memory (Dani and Harris, 2005; Gotti and Clementi, 2004; Steinlein et al., 2012). Identifying the mechanisms responsible for this patterning will provide insights into how behaviors are readily adaptable with a constantly changing environment. The ligand-gated ion channel family is broadly expressed throughout the nervous system and is a major contributor to neuronal patterning (Doyle, 2004; Lindstrom, 2003). While many of the receptor families and

individual subunits have been identified, the functional consequences for heterogeneity among individual receptor families remain unclear. Functional diversity remains difficult to evaluate directly due to the complexity of neuronal connectivity patterns and the uncertainty about the molecular composition of specific receptor types in vivo. Differences in subunit composition have been linked to changes in localization and kinetic properties of the receptors (Glykys and Mody, 2007; Imoto et al., 1988; Revah et al., 1991; Teichert et al., 2012; Zhang et al., 2007b). These changes can have profound effects on neuronal activity. Therefore, understanding how molecular diversity among individual receptor families gives rise to functional diversity and alters neuronal activity will be important for understanding normal brain physiology and the pathophysiology of disorders that affect post-synaptic receptors.

In C. elegans, excitatory cholinergic motor neurons (ACh MNs) make synaptic contacts onto both muscle cells and GABA MNs that, in turn, make

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31 inhibitory synaptic contacts onto opposing musculature (Figure 2-3A) (White et al., 1986). Proper function of this circuit produces temporally coordinated and balanced excitatory and inhibitory signals that pattern movement. While the anatomical connectivity of this circuit has been well characterized, the signaling mechanisms that underlie coordinated motor neuron activity are not well

understood. Expression studies have revealed that many of the 29 ionotropic acetylcholine receptor subunits encoded by the C. elegans genome are expressed in motor neurons, suggesting that cholinergic signaling may play a prominent role (Cinar et al., 2005; Fox et al., 2005; Jones et al., 2007; Rand, 2007). For example, cholinergic motor neurons express a class of heteromeric acetylcholine-gated ion channel complexes known as ACR-2R (Barbagallo et al., 2010; Jospin et al., 2009). ACR-2Rs are ionotropic receptors of the nicotinic acetylcholine receptor superfamily composed of five distinct subunits (ACR-2, ACR-3, UNC-38, UNC-63 and ACR-12), each of which is essential for function in heterologous expression studies. Loss of ACR-2R leads to relatively subtle changes in behavior; however, gain-of-function acr-2 mutations (acr-2(gf)) have profound consequences including hyperactivation and, in extreme cases, death of ACh MNs (Barbagallo et al., 2010; Jospin et al., 2009). In a forward genetic screen to identify mutations that suppressed the toxic effects of acr-2(gf) (ACR-2L9'S), we isolated several loss-of-function alleles of a partnering acetylcholine receptor subunit, acr-12 (Barbagallo et al., 2010). Specific expression of acr-12 in

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32 ACh MNs of animals coexpressing acr-2(gf) restored toxicity in acr-12 mutants, demonstrating a cell autonomous role for ACR-12 in these neurons.

Here we identify and characterize a second class of ACR-12 containing receptors in GABA motor neurons (ACR-12R*). Animals that have lost GABA ACR-12Rs in GABA motor neurons exhibit inconsistency in body bend amplitude across consecutive body bends. Subcellular localization of GABA neurons ACR-12R show discrete sites of green fluorescent protein (GFP) fluorescence. Using electrophysiology we show loss of acr-12 results in significant loss of inhibitory post synaptic events in the muscle that can be rescued with cell specific

expression of ACR-12 in GABA neurons. Lastly, we demonstrate under conditions of elevated ACh release that acr-12 mutants can suppress the paralyzing effects of channelrhodopsin in upstream cholinergic motor neurons. Taken together our results provide valuable insights into the mechanisms of cholinergic signaling in nervous system function.

MATERIALS AND METHODS

C. elegans strains

C. elegans strains were grown under standard laboratory conditions at 22°C. All strains are derivatives of the N2 Bristol strain (wild type). Transgenic strains were obtained by microinjection to achieve germline transformation. Multiple

independent extragenic lines were obtained for each transgenic strain and data presented are from a single representative transgenic line unless noted

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33 rescuing plasmid (pL15Ek; 30ng/ul) and one or more of the following plasmids: pPRB5 [Punc-47::mCherry], pPRB6 2::mCherry], pPRB47

[Pacr-2::mCherry-RAB-3], pPRB53 [ACR-12-GFPICL], pPRB77

[Pacr-2::ACR-12-GFPICL], pHP7 [Punc-47::ACR-12-GFPICL], pAG21 [ACR-12-GFPC-term]. Stably

integrated lines were generated by X-ray integration and outcrossed at least four times to wild type. The following strains were used in this study: IZ914: 12(ok367)X; IZ853: 12(ok367)X;ufIs57[ACR-12-GFPICL]; IZ984:

acr-12(ok367);ufIs78[Pacr-2::ACR-12::GFPICL]; IZ556: acr-12(ok367);ufIs92[P47::ACR-12-GFPICL]; IZ941: 49(e382)III;acr-12(ok367)X; IZ33:

unc-29(x29);acr-16(ok789); IZ514: unc-unc-29(x29);acr-16(ok789);acr-12(ok367); IZ519: acr-12(ok367);ufIs23[Pacr-2::ChR2-GFP]; IZ801: ufIs23[Pacr-2::ChR2-GFP]; IZ629: ufIs38[ACR-12-GFPC-term];ufIs34[Punc-47::mCherry]; IZ651: ufIs38[ACR-12-GFPC-term];ufIs43[Pacr-2::mCherry]; IZ632:

ufIs26[Punc-4::mCherry];ufIs38[ACR-12-GFPC-term]; IZ557: ufIs63[Pacr-2::mCherry-RAB-3];ufIs92[Punc-47::ACR-12-GFPICL]; CB382: unc-49(e382)III; IZ712: acr-12(uf77); RB1559: 2(ok1887)II; IZ805: ufIs53[Punc-17::ChR2-mCherry]; IZ1096: 2(ok1887);ufIs23[P2::ChR2-GFP]; and IZ1095:

acr-12(ok367);ufIs53[Punc17::ChR2-mCherry].

Molecular Biology

The ACR-12-GFPICL transgene (pPRB53) was generated by cloning the GFP

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(-34 1514 to +4799 bp relative to the translational start site) encoding the intracellular loop (ICL) between transmembrane domains (TM)3 and TM4. ACR-12-GFPICL

was localized in neuronal processes and expression of this construct was

sufficient for rescue of acr-12 mutants. pAG21 [ACR-12-GFPC-term] was provided

by Alexander Gottschalk and was used in cell identifications (Fig. 1). ACR-12-GFPC-term fluorescence was largely confined to cell bodies.

Punc-47::ACR-12-GFPICL (pHP7) and Punc-47::ACR-12-GFPICL (pPRB77) were generated by

subcloning a 4.9 kb NruI/BglI fragment containing GFP from pPRB53 into

constructs encoding the acr-12 cDNA under control of the appropriate promoters (pBB25 and pHP3 respectively). Punc-47::mCherry (pPRB5) was generated by subcloning mCherry coding sequence downstream of a 1.3 kb promoter for the unc-47 gene. Pacr-2::mCherry (pPRB6) was generated by subcloning a 3.3 kb promoter for the acr-2 gene upstream of the mCherry coding sequence.

Microscopy

Confocal microscopy was performed using a Zeiss Axioskop 2 microscope system and LSM Pascal 5 imaging software (Zeiss). All images used animals 24 h after the L4 stage and were processed using ImageJ software. For all synapse quantification, a region of the dorsal cord directly across from the vulva was imaged. Synapses were quantified within a 50 μM region of interest by

thresholding fluorescence intensity and using the “analyze particles” module in ImageJ software. The percentage of overlap with pre-synaptic RAB-3 was

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35 determined by quantifying the number of mCherry::RAB-3 puncta which were positioned either directly apposing or overlapping with ACR-12::GFP fluorescent signal.

Behavioral assays

All behavioral analyses were performed using staged populations of young adult animals (24 h following L4) at room temperature (22°C-24°C). Strains were scored in parallel, with the researcher blinded to the genotype during both experiment and analysis. For aldicarb assays, staged populations of adult animals (≥10) were transferred to NGM plates containing 1 mM aldicarb

(ChemService), and movement was assessed every 15 min for 2 h. Movies and still images for behavioral analyses were obtained using an Olympus SZ61

upright microscope equipped with a FireWire camera (Imaging Source). For body bend measurements, amplitudes were determined using ImageJ software. The distance between the deepest point of the bend and a line tangent to the tip of the head and the body was measured. This measurement was then normalized to the length of each animal and either averaged across 3 consecutive body bends to generate a value for average body bend amplitude (Figure 2-6D) or 10 individual consecutive body bends (Figure 2-6B and C). For gross movement, individual worms were transferred to 100mm (large) unseeded plates. The number of body bends and spontaneous reversals were counted manually over 1-minute intervals for 3-minutes and averaged (Figure 2-6E). For optogenetic

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36 experiments, animals were maintained on OP50 plates containing retinal and 450-490 nm light (2.5 mW/mm2) was delivered using an Exfo X-cite series 120 light source and appropriate GFP excitation filters. On the day of the assay staged young adult animals were moved to a fresh plate for an equilibration period of 1 minute prior to filming for 40 s. After 10 s of filming the animals were exposed to blue light for 15 s and then filmed for an additional 10 s after blue light exposure. Responses were categorized using the following criteria: no movement impairment, tail bend ≤ 90°, tail bend > 90°, or arrested movement.

Electrophysiology

Endogenous postsynaptic currents were recorded from body wall muscles as previously described (Francis et al., 2005). The extracellular solution

consisted of 150 mM NaCl, 5 mM KCl, 4 mM MgCl2, 1 mM CaCl2, 15 mM

HEPES, and 10 mM glucose (pH 7.4, osmolarity adjusted with 20 mM sucrose). The intracellular fluid (ICF) consisted of 115 mM K-gluconate, 25 mM KCl, 0.1 mM CaCl2, 50 mM HEPES, 5 mM Mg-ATP, 0.5 mM Na-GTP, 0.5 mM cGMP, 0.5

mM cAMP, and 1 mM BAPTA (pH 7.4, osmolarity adjusted with 10 mM sucrose). For some experiments measuring GABA-mediated currents, the intracellular solution contained 115 mM KCl and 25 mM K-gluconate. At least 60-90 s of continuous data were used in the analysis. Data analysis was performed using Igor Pro (WaveMetrics, Inc.) and Mini Analysis (Synaptosoft, Inc.) software. Statistical comparisons were made by Student’s t test using GraphPad Prism.

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37

RESULTS

Loss of ACR-12 leads to heightened excitability

acr-12 encodes a 573 amino acid iAChR alpha subunit (Figure 1) with significant homology to the ACR-8 like group of C. elegans subunits and more distant homology to mammalian neuronal heteromeric alpha subunits such as alpha6 (Jones et al., 2007). To investigate the contribution of ACR-12 receptor complexes to the excitability of neurons in the motor circuit, we evaluated independent strains carrying putative loss of function alleles of acr-12 during acute exposure to the cholinesterase inhibitor aldicarb. Treatment of C. elegans with aldicarb leads to elevated levels of synaptic ACh and causes paralysis over time due to prolonged muscle contraction (Nguyen et al., 1995). The contractile state of muscles reflects the summed activity of excitatory ACh and inhibitory GABA synaptic inputs; genetic mutations that alter this balance will shift the time course over which aldicarb leads to paralysis. For example, manipulations that reduce or eliminate inhibitory GABA signaling cause enhanced muscle activation and more rapid paralysis (Loria et al., 2004; Vashlishan et al., 2008).

acr-12(uf77) was isolated as a suppressor of acr-2(gf) and is a missense mutation affecting a splice acceptor site that results in premature termination prior to transmembrane domain 4 (aa 387) (Barbagallo et al., 2010). acr-12(ok367) is a deletion mutation that eliminates 1368 bp of chromosomal DNA, including sequence encoding transmembrane domains 1-3 (Figure 2-1). Strains carrying either of these alleles were viable and gross connectivity of the nervous system

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38 was normal (not shown), indicating that ACR-12 is not required for normal

development of the nervous system. We found the 12(uf77) and acr-12(ok367) mutations both accelerated the time course of aldicarb-induced paralysis (Figure 2-2A), suggesting loss of ACR-12 led to enhanced excitability. In contrast, previous work showed removal of ACR-2Rs slightly delayed paralysis in response to aldicarb treatment (Barbagallo et al., 2010; Jospin et al., 2009). The differential effects of aldicarb across acr-2 and acr-12 mutants suggested ACR-12 has additional roles in the nervous system independent of ACR-2. To better define potential functions for ACR-12 signaling, we constructed double mutants lacking both acr-12 and unc-49. The unc-49 gene encodes an essential subunit of ionotropic GABAA-like receptors at inhibitory neuromuscular synapses

and mutations in unc-49 cause aldicarb hypersensitivity (Figure 2-2A) (Bamber et al., 1999; Vashlishan et al., 2008). unc-49;acr-12 double mutants exhibited no additional hypersensitivity beyond that of unc-49 single mutants, suggesting that acr-12 and unc-49 may act in the same pathway.

acr-12 is reported to have broad expression in the nervous system (Gottschalk et al., 2005) but a precise description of the motor neuron classes that express acr-12 has remained unclear. To address this issue, we generated transgenic strains expressing ACR-12 tagged with GFP (green fluorescent protein) at the C-terminus together with a red fluorescent reporter

(Pacr-2::mCherry) labeling ACh motor neurons, and examined the cellular distribution of ACR-12-GFPin the ventral nerve cord. ACR-12-GFP fluorescence was

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39 broadly visible in motor neuron cell bodies and partially overlapped with Pacr-2::mCherry expression, confirming that acr-12 is expressed in cholinergic motor neurons (Figure 2-2D). In addition we coexpressed ACR-12-GFP with a red fluorescent protein reporter for the A class and VC motor neurons

(Punc-4::mCherry) and observed a partial overlap of ACR-12-GFP that did not include the VC neurons (Figure 2-2E). Fluorescence was also clearly visible in additional motor neurons along the ventral cord. To confirm the identity of these neurons, we imaged animals co-expressing ACR-12-GFP with a reporter labeling GABA neurons (Punc-47::mCherry) and observed overlapping fluorescent signals (Figure 2-2F). Our results indicate ACR-12 contributes to iAChRs expressed by both ACh and GABA motor neurons. Furthermore, the limited expression of some ACR-2R constituents to ACh MNs strongly suggests the iAChR

populations expressed by ACh and GABA motor neurons are molecularly distinct (Barbagallo et al., 2010; Jospin et al., 2009).

To dissect functional roles for ACR-12 across motor neuron classes, we specifically expressed ACR-12 in either ACh or GABA motor neurons of acr-12 mutants and tested the responses of these animals to aldicarb treatment (Figure 2-2B). Specific expression of acr-12 in ACh MNs did not alter the aldicarb

hypersensitivity and paralysis of acr-12 mutants. In contrast, specific expression of acr-12 in GABA neurons restored wild type sensitivity to aldicarb. Our results suggest that postsynaptic ACR-12 receptor complexes regulate GABA motor

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40 neuron activity and inhibitory neuromuscular signaling under conditions when ACh levels are elevated.

iAChRs requiring ACR-12 have distinct patterns of localization and

functional roles across motor neuron classes

We examined the subcellular distribution of ACR-12 in motor neurons by expressing a rescuing ACR-12-GFP fusion protein under control of the native promoter (Figure 3B). ACR-12-GFP expression was clearly visible in motor neuron processes of both the ventral and dorsal nerve cords of adult animals, and exhibited two contrasting patterns of fluorescence. In the ventral nerve cord we observed regions of punctate and diffuse localization while, in the dorsal cord, we observed punctate fluorescence almost exclusively (Figure 2-3B). In contrast, specific expression of ACR-12-GFP in ACh MNs produced only diffuse

fluorescence in the ventral nerve cord and no detectable fluorescent signal in the dorsal nerve cord (Figure 2-3C). Axons and dendrites of ventrally directed

cholinergic motor neurons (ventral A and B classes) extend through the ventral nerve cord. Cholinergic motor neurons innervating dorsal musculature (dorsal A and B classes) extend axons into the dorsal nerve cord, where they make dyadic synaptic contacts with body wall musculature and dendrites of ventrally directed GABA motor neurons (ventral D class) (Figure 2-3A) (White et al., 1986). The diffuse localization of ACR-12 in ACh MNs is consistent with our previous finding that ACR-12 contributes to heteromeric receptor complexes (e.g. ACR-2R)

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41 without obvious postsynaptic localization in these neurons (Barbagallo et al., 2010). Specific expression of ACR-12-GFP in GABA MNs produced solely punctate fluorescence (Figure 2-3D). Many of these ACR-12-GFP puncta were located immediately opposed to regions of cholinergic motor neuron axons in which the synaptic vesicle marker mCherry-RAB-3 was concentrated (Figure 2-4A-C) (Klassen and Shen, 2007; Mahoney et al., 2006). Our results provide evidence that ACR-12 complexes in GABA MNs are clustered in receptor fields located opposite presynaptic specializations of ACh MNs.

GABA motor neuron expression of ACR-12 is required for normal levels of

inhibitory synaptic activity

To directly test the requirement for ACR-12 in regulating motor neuron activity, we used standard electrophysiology recording techniques to measure the frequency of synaptic events at the NMJ in vivo (Figure 2-5) (Francis and Maricq, 2006). We employed two independent strategies to distinguish between GABA and ACh post synaptic currents (PSCs): 1) Recordings were made under ionic conditions where GABA and ACh mediated events were separable based on the directionality of the currents (see Experimental Procedures for details) and 2) We recorded from unc-29;acr-16 double mutants that are devoid of functional iAChRs on body wall muscles and lack excitatory neurotransmission at the NMJ [18, 19]. In both cases, we observed a significant reduction in the rate of

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42 receptors (53%, p<0.001 and 69 %, p<0.01 respectively) (Figure 2-5). We did not observe a significant difference in the amplitude of endogenous inhibitory PSCs (wild type, 26 ± 1.8 pA; acr-12, 23.4 ± 2 pA, p>0.05, Figure 5). To distinguish whether the reduction in inhibitory PSC frequency arose as a consequence of loss of ACR-12 from GABA MNs or arose due to reduced excitation levels in presynaptic ACh MNs, we recorded from animals that expressed acr-12 specifically in ACh or GABA MNs (Figure 2-5). The reduced IPSC rate was partially rescued by expression of acr-12 in GABA motor neurons, while expression in ACh MNs was not sufficient for rescue. Additionally, the acr-12 deletion mutation did not significantly reduce the rate or amplitude of

endogenous excitatory PSCs (wild type, 28.6 ± 1.8 pA; acr-12, 26.8 ± 1.3 pA, p>0.05), although we did note a trend toward reduced frequency (33%, p = 0.07) (Figure 2-5E). Thus, the reduction in endogenous IPSC frequency cannot be explained by decreased excitation of presynaptic ACh MNs. Our results indicate that independent populations of ACR-12 receptors act cell autonomously in ACh and GABA MNs to regulate their activity.

ACR-12 mediated signaling onto motor neurons regulates locomotion

To determine how ACR-12 mediated signaling onto motor neurons contributes to normal locomotory behavior, we tracked animals during exploratory movements on agar plates. The sinusoidal locomotory wave

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43 detail, we monitored consecutive body bends during extended periods of

uninterrupted forward movement (Figure 2-6A and B). Wild type animals

displayed remarkable consistency in their movement with only a few irregularities in their sinusoidal tracks. In the absence of ACR-12 receptors (ok367 and uf77), the motor pattern was less stable, showing significantly increased variability in body bend amplitude from one body bend to the next (Figure 2-6C, p<0.001). Furthermore, acr-2(ok1887) mutants do not have an irregular waveform

suggesting loss of GABA-specific ACR-12 iAChRs is required for coordination of a consistent waveform. Animals expressing a genomic ACR-12-GFP reduced variability to wild type levels (Figure 2-6C green). In addition to an inconsistent waveform we observe a modest reduction in the average amplitude of body bends (~14% for both uf77 and ok367, p<0.05 and p<0.01 respectively) (Figure 2-6D). Body bend amplitude was reversed by ACR-12 expression using the native promoter. In addition, specific expression of ACR-12 in GABA motor neurons, but not cholinergic motor neurons reversed the reduction in body bend amplitude. Moreover, acr-2(ok1887) mutants did not exhibit changes in body bend amplitude. These disruptions in body bend variability and amplitude likely contribute to the overall decrease acr-12(ok367) mutant movement (Figure 2-6E). In addition to changes in the number of body bends over time acr-12(ok367) mutants also exhibit an increase in the frequency of spontaneous reversals (Figure 2-6E). This increase may indicate important roles for ACR-12 iAChRs in the interneurons. Here we highlight how ACR-12 receptors under laboratory

References

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